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Maria Belland Olsen,1,2 Camilla Huse,1,2 Mirta Mittelstedt Leal de Sousa,3,4 Sarah Louise Murphy,1,2 Antonio Sarno,3,5 Tobias Sebastian Obermann,3 Kuan Yang,1 Jan Cato Holter,2,6 Marte Jøntvedt Jørgensen,2,7 Erik Egeland Christensen,2,7 Wei Wang,3 Ping Ji,3 Lars Heggelund,8,9 Hedda Hoel,1,10 Anne Margarita Dyrhol-Riise,2,7 Ida Gregersen,1 Pål Aukrust,1,2,11 Magnar Bjørås,3,6 Bente Halvorsen,1,2 Tuva Børresdatter Dahl12 1Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway; 2Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 3Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway; 4Proteomics and Modomics Experimental Core Facility (PROMEC), NTNU, Trondheim, Norway; 5Department of Fisheries and New Biomarine Industry, SINTEF Ocean, Trondheim, Norway; 6Department of Microbiology, Oslo University Hospital and University of Oslo, Oslo, Norway; 7Department of Infectious Diseases, Oslo University Hospital, Oslo, Norway; 8Department of Internal Medicine, Vestre Viken Hospital Trust, Drammen, Norway; 9Department of Clinical Science, Faculty of Medicine, University of Bergen, Bergen, Norway; 10Department of Medicine, Lovisenberg Diaconal Hospital, Oslo, Norway; 11Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital, Oslo, Norway; 12Division of Critical Care and Emergencies, Oslo University Hospital, Oslo, NorwayCorrespondence: Tuva Børresdatter Dahl, Division of Critical Care and Emergencies and Research Institute of Internal Medicine, Oslo University Hospital, Sognsvannsveien 20, Oslo, Norway, Tel +4723072786, Email t.b.dahl@medisin.uio.noPurpose: Reactive oxygen species (ROS) are an important part of the inflammatory response during infection but can also promote DNA damage. Due to the sustained inflammation in severe Covid-19, we hypothesized that hospitalized Covid-19 patients would be characterized by increased levels of oxidative DNA damage and dysregulation of the DNA repair machinery.Patients and Methods: Levels of the oxidative DNA lesion 8-oxoG and levels of base excision repair (BER) proteins were measured in peripheral blood mononuclear cells (PBMC) from patients (8-oxoG, n = 22; BER, n = 17) and healthy controls (n = 10) (Cohort 1). Gene expression related to DNA repair was investigated in two independent cohorts of hospitalized Covid-19 patients (Cohort 1; 15 patents and 5 controls, Cohort 2; 15 patients and 6 controls), and by publicly available datasets.Results: Patients and healthy controls showed comparable amounts of oxidative DNA damage as assessed by 8-oxoG while levels of several BER proteins were increased in Covid-19 patients, indicating enhanced DNA repair in acute Covid-19 disease. Furthermore, gene expression analysis demonstrated regulation of genes involved in BER and double strand break repair (DSBR) in PBMC of Covid-19 patients and expression level of several DSBR genes correlated with the degree of respiratory failure. Finally, by re-analyzing publicly available data, we found that the pathway Hallmark DNA repair was significantly more regulated in circulating immune cells during Covid-19 compared to influenza virus infection, bacterial pneumonia or acute respiratory infection due to seasonal coronavirus.Conclusion: Although beneficial by protecting against DNA damage, long-term activation of the DNA repair machinery could also contribute to persistent inflammation, potentially through mechanisms such as the induction of cellular senescence. However, further studies that also include measurements of additional markers of DNA damage are required to determine the role and precise molecular mechanisms for DNA repair in SARS-CoV-2 infection.Keywords: Covid-19, oxidative stress, DNA damage, DNA repair, base excision repair, double strand break repair

DNA double-strand breaks (DSBs) trigger the Ataxia telangiectasia mutated (ATM)-dependent DNA damage response (DDR), which consists of histone H2AX, MDC1, RNF168, 53BP1, PTIP, RIF1, Rev7, and Shieldin. Early stages of B and T lymphocyte development are dependent on recombination activating gene (RAG)-induced DSBs that form the basis for further V(D)J recombination. Non-homologous end joining (NHEJ) pathway factors recognize, process, and ligate DSBs. Based on numerous loss-of-function studies, DDR factors were thought to be dispensable for the V(D)J recombination. In particular, mice lacking Mediator of DNA Damage Checkpoint Protein 1 (MDC1) possessed nearly wild-type levels of mature B and T lymphocytes in the spleen, thymus, and bone marrow. NHEJ factor XRCC4-like factor (XLF)/Cernunnos is functionally redundant with ATM, histone H2AX, and p53-binding protein 1 (53BP1) during the lymphocyte development in mice. Here, we genetically inactivated MDC1, XLF, or both MDC1 and XLF in murine vAbl pro-B cell lines and, using chromosomally integrated substrates, demonstrated that MDC1 stimulates the V(D)J recombination in cells lacking XLF. Moreover, combined inactivation of MDC1 and XLF in mice resulted in synthetic lethality. Together, these findings suggest that MDC1 and XLF are functionally redundant during the mouse development, in general, and the V(D)J recombination, in particular. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. The Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factor subunits X-ray repair cross-complementing group 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase 4 (Lig4). Accessory factors might be dispensable for the process, depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced a frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as the wild type controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development. Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).